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At least 19 recordsLinked to original sources
PubMed2006

Differential expression of smooth muscle myosin, smooth muscle actin, h-caldesmon, and calponin in the diagnosis of myofibroblastic and smooth muscle lesions of skin and soft tissue.

The diagnosis of low-grade and pseudosarcomatous spindle cell lesions of skin and soft tissue can sometimes be problematic; in particular, distinction between fibroblastic, myofibroblastic, and smooth muscle proliferations can occasionally pose difficulties on routine histologic examination. We have applied a panel of immunohistochemical markers to a series of spindle cell lesions of skin and soft tissue to assess the utility of the differential expression of smooth muscle and myofibroblastic-associated markers. Twenty-eight cases of nodular fasciitis, 42 cases of fibromatosis, and 3 cases of myofibroblastic sarcoma were stained with antibodies against smooth muscle actin (SMA), smooth muscle myosin (SMMS), calponin, and high-molecular weight caldesmon (h-caldesmon). For comparison, 12 cases of cutaneous leiomyoma and 8 cases of leiomyosarcomas involving superficial soft tissues and fascia were studied with the same panel of antibodies. Thirty-eight of 42 cases of fibromatosis were positive for SMA, 42/42 cases were positive for calponin, 39/42 cases were negative for SMMS, and all cases were negative for h-caldesmon. All cases of nodular fasciitis were positive for SMA and calponin, and all were negative for h-caldesmon and SMMS. All cases of myofibroblastic sarcoma were positive for SMA and 2/3 cases for calponin, and were negative for SMMS and h-caldesmon. All cases of cutaneous leiomyoma and leiomyosarcoma were positive for all 4 markers tested. Our results demonstrate a remarkably consistent pattern of reactivity of muscle and myofibroblastic-associated markers in lesions predominantly composed of myofibroblastic spindle cells, characterized by positive staining for SMA and calponin and absence of reactivity for SMMS and h-caldesmon. Application of this panel of stains may be of aid in the differential diagnosis of low-grade myofibroblastic lesions such as nodular fasciitis and fibromatosis from smooth muscle tumors of skin and soft tissue. This panel may additionally be of assistance in the diagnosis of myofibroblastic sarcoma.

Actins↗
PubMed1995

Effect of caldesmon on the assembly of smooth muscle myosin.

Smooth muscle myosin filaments are much less stable than the skeletal muscle counterpart. Smooth myosin requires higher concentration of Mg2+ than skeletal myosin to form thick filaments and addition of ATP disassembles the dephosphorylated smooth muscle myosin filaments into monomers but not phosphorylated ones. We found that the addition of caldesmon to dephosphorylated myosin induced the formation of the filaments under the conditions where myosin by itself is soluble or disassembled. Although the induced filaments were short at 1 mM Mg2+, they became medium sized and seemed like side polar filaments with prominent 14 nm periodicity at higher Mg2+ conditions (8 mM). In the presence of F-actin, myosin filaments induced by caldesmon were associated along actin filaments to form large structures. The association of actin and myosin filaments was observed only in the presence of caldesmon, suggesting that caldesmon cross-linked actin and myosin filaments. This cross-linking was disrupted by the addition of calmodulin. Caldesmon-induced filament formation of dephosphorylated myosin in the presence of Mg(2+)-ATP may explain the existence of myosin filaments in relaxed smooth muscle fibers. A similar effect of telokin on myosin filament assembly was also examined and is discussed.

Actins↗
PubMed1998

Regulation and tuning of smooth muscle myosin.

Smooth muscle myosin is regulated by phosphorylation of one of the two myosin light chains. This phosphorylation causes an unfolding of the myosin that allows it to interact with actin to produce force. The inactive state involves trapping the myosin in a conformation wherein the myosin heads interact with a segment of the myosin rod. Phosphorylation of the regulatory light chain weakens these interactions and allows the myosin to be activated. Smooth muscle myosin has a large movement of its light chain binding domain that is coupled to ADP release. This structural change may be necessary for the generation of "latch." Smooth muscle myosin has three different regions that vary to generate different isoforms: (1) an alternative insertion within the myosin head; (2) two possible essential light chains; and (3) an alternative tail at the end of the myosin rod. There is substantial evidence that the insertion in the myosin head increases the enzymatic activity of the myosin and leads to greater shortening velocity. The function of the other two variants is as yet unclear.

Actins↗
PubMed1998

The molecular mechanics of smooth muscle myosin.

Smooth muscle cells are capable of generating forces comparable to those of skeletal muscle cells but with far less myosin, the molecular motor that powers muscle contraction. This unique capability may be inherent to the myosin molecule. We have directly characterized the molecular mechanics of smooth muscle myosin using new technologies developed to measure the forces generated by these proteins. The data help explain the differences in force and velocity in whole smooth and skeletal muscles.

Animals↗
PubMed2004

Novel sensors of the regulatory switch on the regulatory light chain of smooth muscle Myosin.

Smooth muscle myosin can be switched on by phosphorylation of Ser-19 of the regulatory light chain. Our previous photocross-linking results suggested that an element of the structural mechanism for the regulatory switch was a phosphorylation-induced motion of the regulatory light chain N terminus (Wahlstrom, J. L., Randall, M. A., Jr., Lawson, J. D., Lyons, D. E., Siems, W. F., Crouch, G. J., Barr, R., Facemyer, K. C., and Cremo, C. R. (2003) J. Biol. Chem. 278, 5123-5131). Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.

2-Naphthylamine↗
PubMed1988

Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin.

Smooth muscle myosin can be phosphorylated by myosin light chain kinase at the serine 19 and threonine 18 residues of the two 20,000-dalton light chains (Ikebe, M., Hartshorne, D. J., and Elizinga, M. (1986) J. Biol. Chem. 261, 36-39). These studies with myosin and heavy meromyosin (HMM) compare the effects induced by phosphorylation of serine 19 (M2P and HMM2P) and serine 19 plus threonine 18 (M4P and HMM4P). Formation of M4P altered the KCl dependence of viscosity and Mg2+-ATPase and higher values were maintained at lower ionic strengths, compared to M2P or dephosphorylated myosin (Mo). This is consistent with the stabilization of the 6 S conformation. The tendency for aggregation, as judged by light scattering, followed the sequence M4P greater than M2P greater than Mo. Filaments formed with M4P were more resistant to dissociation by ATP compared to filaments of M2P. Phosphorylation of HMM2P doubled Vmax of actin-activated ATPase with little effect on the apparent affinity for actin. The Mg2+-ATPase of HMM4P exhibited a higher activity at low ionic strength compared to HMM2P and HMMo. Hydrodynamic differences were detected at low ionic strength in the presence of ATP by sedimentation velocity measurements with HMM4P, HMM2P, and HMMo. Proteolysis by papain indicated an increased susceptibility of the head-neck junction of HMM4P compared to HMM2P. These data suggest that the phosphorylation of threonine 18 in addition to serine 19 change the conformation of myosin and HMM and this is associated with altered biological properties.

Animals↗
PubMed2001

Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin.

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.

Animals↗
PubMed1981

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin.

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.

Adenosine Triphosphatases↗
PubMed1995

Role of ATP in the binding of caldesmon to smooth muscle myosin.

We have reported earlier that ATP causes both an increase in the affinity of caldesmon for smooth muscle myosin and a change in stoichiometry from 2 caldesmon molecules per myosin to 1:1 (Hemric & Chalovich, 1990). We now show that this ATP effect does not occur with skeletal muscle myosin, indicating that ATP has a specific effect on the structure of filamentous smooth muscle myosin. This ATP effect does not appear to be due to stabilization of a 10S type of filamentous smooth muscle myosin like that reported earlier (Ikebe & Hartshorne, 1984) since neither phosphorylation nor extensive modification of myosin with MalNEt (both which stabilize the 6S state of monomeric myosin) eliminates the effect of ATP. Caldesmon does bind more tightly to a form of smooth muscle myosin which is resistant to papain digestion. These results suggest that the ATP effect is due to stabilization of a local conformation of smooth muscle myosin which is independent of the larger 10S/6S conformational change (Suzuki et al., 1988). In the presence of ATP, the two heads of smooth muscle muscle myosin and the S-2 region form a single, higher affinity binding region for caldesmon.

Adenosine Diphosphate↗
PubMed1997

An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms.

Smooth muscle myosin isoforms of the heavy chain and the essential light chain have been hypothesized to contribute to the different shortening velocities of phasic and tonic smooth muscles, and to their different affinities for MgADP. We used the baculovirus/insect cell system to express homogeneous heavy meromyosin molecules differing only in seven amino acid insert (QGPSFSY) in the motor domain near the active site, or in the type of essential light chain isoform. Myosin from tonic rabbit uterine smooth muscle lacks the heavy chain insert, while myosin from phasic chicken gizzard contains it. The properties of a mutant uterine heavy meromyosin with added insert, and a mutant gizzard heavy meromyosin with the insert deleted, were compared with their wild type progenitors. Phosphorylated heavy meromyosins with the insert have a twofold higher enzymatic activity and in vitro motility han heavy meromyosins without the insert. These functional properties were not altered by the essential light chain isoforms. The altered motility caused by the insert implies that it modulates the rate of ADP release, the molecular step believed to limit shortening velocity. The insert may thus account in part for both the lower sensitivity to MgADP and the higher shortening velocity of phasic compared to tonic smooth muscles.

Actins↗
PubMed1996

Structure and function of the 10 S conformation of smooth muscle myosin.

Smooth myosin regulatory light chain (RLC) was exchanged with RLC labeled with benzophenone-4-iodoacetamide at Cys-108. Irradiation under conditions that favor the folded (10 S) conformation resulted in 10 S cross-linked myosin that could not unfold. Purified 10 S cross-linked myosin was cross-linked between the RLC of one head to light meromyosin between leucine 1554 and glutamate 1583, adjacent to a predicted noncoiled region, approximately 60 nm from the tip of the tail. At high ionic strength without actin, product release from one-half of the heads was slow (like 10 S) whereas the other half were activated. This suggests that tail binding to the RLC carboxyl-terminal domain stabilizes ionic interactions important to slow nucleotide release. With actin, product release from both (un)phosphorylated 10 S cross-linked myosin was from one slow population similar to unphosphorylated filaments. 10 S cross-linked myosin weakly bound actin (dissociation constant > 500 microM) and did not move actin in vitro. Single-headed myosin did not fold or trap nucleotide. These and other data suggest that "trapping" occurs only with both heads and the tail binds to a newly formed site, which includes the RLC carboxyl-terminal domain, once trapping has occurred.

Actins↗
PubMed1995

Smooth muscle myosin II and alpha smooth muscle actin expression in the baboon (Papio anubis) uterus is associated with glandular secretory activity and stromal cell transformation.

The objective of this study was to investigate the localization and hormonal regulation of smooth muscle myosin II (SMM II) and alpha smooth muscle actin (alpha SMA) in the baboon uterus, since cytoskeletal proteins are involved in secretory function and morphological transformation. Uterine tissue was obtained from baboons 1) during the menstrual cycle, 2) following steroid treatment of ovariectomized baboons, 3) during pregnancy (Days 14-60 postovulation [PO]), and 4) during simulated pregnancy (Days 18-32 PO). Tissues were processed for immunocytochemical localization of SMM II or alpha SMA with specific polyclonal or monoclonal antibodies, respectively. SMM II stained all smooth muscle cells of blood vessels and myometrium regardless of treatment. Glandular epithelial staining was present only in endometrium obtained during the luteal phase or following estrogen and progesterone treatment. Staining intensity was greater in the basalis than in the functionalis. The number of glands staining positive for SMM II on Days 18-32 of pregnancy and simulated pregnancy was variable. Glandular stain was absent after Day 32 PO. These immunocytochemical data were confirmed by immunoblot analysis of glandular cytosolic extracts. Stromal staining for SMM II was present under the luminal epithelium during simulated pregnancy (Days 18-32), on Day 25 of steroid treatment in the simulated-pregnant controls, and in nonimplantation sites during pregnancy. In contrast, alpha SMA staining was low or absent in all uterine cell types in ovariectomized baboons. Under estrogen-dominated conditions (follicular phase and estrogen treatment), alpha SMA staining was present in smooth muscle cells, and this staining persisted throughout the remaining treatment periods. Glandular epithelial staining for alpha SMA was absent in all treatment groups. However, alpha SMA staining in stromal fibroblasts underneath the luminal epithelium was evident as early as Day 14 of pregnancy and Day 18 of simulated pregnancy. The number of stromal fibroblasts that stained positive increased in the surface region of the functionalis between Days 18 and 32 PO, and the staining extended throughout the upper functionalis region. There was a decrease in the number of positively stained stromal fibroblasts, particularly at the implantation site, between Days 32 and 40 of pregnancy. By Days 50-60 of pregnancy, this staining was almost absent. The induction of alpha SMA in stromal fibroblasts in the functionalis region in pregnant baboons was confirmed by immunoblot analysis of stromal cell cytosol extracts. We conclude that the progesterone-induced glandular expression of SMM II may be involved in uterine secretory function and that alpha SMA expression in stromal fibroblasts during pregnancy and after long-term steroid treatment is associated with the decidualization process.

Actins↗
PubMed1992

Role of 17-kDa essential light chain isoforms of aorta smooth muscle myosin.

Aorta smooth muscle myosin contains two kinds of 17-kDa essential light chain, LC17nm (nonmuscle-type) and LC17gi (gizzard-type) [Hasegawa, Y., Ueda, Y., Watanabe, M., & Morita, F. (1992) J. Biochem. 111, 798-803]. The LC17 isoforms were released from porcine aorta myosin by incubation at 46 degrees C. The rate of release was 1.5 to 2 times higher with LC17gi than with LC17nm. Aorta myosins containing the two LC17 isoforms in various ratios could be reconstituted. The actin-activated ATPase activity was measured as a function of LC17nm content. The Vm value was lower with myosin which contained more LC17nm. The apparent dissociation constant for F-actin, Km, was 20-fold less with myosin which contained 81% LC17nm than myosin which contained 23% LC17nm. A similar difference in the dissociation constants of myosin for F-actin was observed in the presence of adenylyl imidodiphosphate. The role of LC17nm appears to be to make aorta myosin suitable for maintaining the muscle tension with a low expenditure of energy. The isoform-dependent difference in the F-actin-binding affinities of myosin seems partly due to the difference in the affinities of LC17 isoforms themselves for F-actin. We found that the isolated LC17nm itself could bind with F-actin with a dissociation constant of 64 microM, but LC17gi could not.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed2000

Diagnostic implications of elevated levels of smooth-muscle myosin heavy-chain protein in acute aortic dissection. The smooth muscle myosin heavy chain study.

BACKGROUND: A rapid 30-minute assay of circulating smooth-muscle myosin heavy-chain protein has been developed as a biochemical diagnostic tool for aortic dissection. OBJECTIVE: To determine the sensitivity and specificity of this assay. DESIGN: Cross-sectional study. SETTING: 8 major cardiovascular centers in Japan. PATIENTS: 95 patients with acute aortic dissection, 48 patients with acute myocardial infarction, and 131 healthy volunteers. MEASUREMENTS: Levels of circulating smooth-muscle myosin heavy-chain protein. RESULTS: Patients with acute aortic dissection who presented within 3 hours after onset had elevated levels of circulating smooth-muscle myosin heavy-chain protein. In these patients, the assay had a sensitivity of 90.9%, a specificity of 98% compared with healthy volunteers, and a specificity of 83% compared with patients who had acute myocardial infarction; the clinical decision limit was 2.5 microgram/L. All patients with proximal lesions had elevated levels of smooth-muscle myosin heavy-chain protein, and only patients with distal lesions had decreased levels (<2.5 microgram/L). CONCLUSIONS: Levels of smooth-muscle myosin heavy-chain protein can be used to diagnose aortic dissection soon after symptom onset. The assay had the greatest diagnostic value in patients with proximal lesions.

Aged↗
PubMed1995

Expression of smooth muscle myosin isoforms in urinary bladder smooth muscle during hypertrophy and regression.

BACKGROUND: Partial ligation of the urinary out-flow tract of rabbit bladder induces hypertrophy of the smooth muscle layer in the bladder wall, and it is reversible by the removal of the ligature. The expression of smooth muscle myosin heavy chain isoforms SM1 and SM2 after hypertrophy and the regression of hypertrophy (reversal) was investigated at the translational and transcriptional levels using this experimental model. DESIGN: The contractile activity of smooth muscle strips derived from normal, hypertrophied, and reversal bladders was measured using electrical stimulation. Expression of SM1 and SM2 in normal, hypertrophied, and reversal muscle tissue was characterized using SDS-PAGE, reverse transcriptase-PCR (RT-PCR), and RNase protection assay. RESULTS: Smooth muscle strips from hypertrophied urinary bladder revealed a decrease in both force and rate of force generation in response to field stimulation. These alterations in contractility were reversed by removal of the obstruction. The altered function in bladder hypertrophy was also associated with changes in translation and transcription of the smooth muscle heavy chain isoforms SM1 and SM2. Upon regression of the hypertrophy by removal of the obstruction, the relative ratio of myosin heavy chain SM2:SM1 returned to nearly normal values. Analyses by RT-PCR showed a decrease in the mRNA transcript for SM2 in hypertrophied bladder muscle; and, on reversal of the hypertrophy, the SM2 mRNA level returned to that of normal bladder. These data suggest that the obstruction-induced hypertrophy activates a down-regulating mechanism for the expression of myosin SM2 heavy chain. CONCLUSIONS: Obstruction-induced alteration in the contractile characteristics of the urinary bladder smooth muscle is associated with changes in the expression of smooth muscle myosin heavy chains at both the protein and mRNA levels. The contractile function and the myosin heavy chain expression return to normal after regression of the smooth muscle hypertrophy on removal of the obstruction.

Amino Acid Sequence↗
PubMed1988

The heavy-chain stoichiometry of smooth muscle myosin is a characteristic of smooth muscle tissues.

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.

Animals↗